Microbial pathogens have developed a variety of strategies to infect their human host and cause disease. Many Gram-negative bacteria use type IV secretion systems (T4SSs) to deliver bacterial proteins, called effectors, into host cells. The effectors help to modulate signaling events within the host in order to create conditions favorable for bacterial survival. We are committed to the in-depth analysis of microbial virulence strategies. We use as a model organism the bacterium Legionella pneumophila, the causative agent of a potentially fatal respiratory infection known as Legionnaires' disease. Each year more individuals in the U.S. contract Legionnaires' disease (8,000 to 18,000) than there are cases of ALS (Amyotrophic Lateral Sclerosis or Lou Gehrig's Disease), thus making L. pneumophila a significant health threat and a considerable economic burden. Moreover, the infection cycle of L. pneumophila shows numerous parallels to the virulence programs of Salmonella, Chlamydia, Mycobacterium, Coxiella, and many other human pathogens that manipulate host cells from within a membrane-enclosed compartment. In addition, given that a type IV secretion system (T4SS), the major virulence apparatus of L. pneumophila, is present in numerous animal and plant pathogens including Helicobacter or Agrobacterium, the in-depth analysis of this translocation system and its cargo proteins, called effectors, is of great importance for our general understanding of microbial virulence. Last but not least, the effector proteins that are used by L. pneumophila to manipulate host cell processes display remarkable parallels to eukaryotic proteins, and deciphering their function will yield valuable insight into mechanistic and regulatory concepts about processes that occur within our own cells. Thus, obtaining a detailed understanding of Legionella's biology and its virulence strategies is essential to more effectively prevent, diagnose, and treat this dangerous pneumonia, and will profoundly improve people's lives and wellbeing. L. pneumophila is ubiquitously found in freshwater habitats such as cooling towers, air conditioning systems, and water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires' disease cases within the U.S. has tripled over the past decade and a half, making this microorganism an emerging public health threat. Upon inhalation, L. pneumophila infects and replicates within alveolar macrophages, specialized immune cells within the human lung. L. pneumophila delivers close to 300 proteins, called effectors, through a T4SS into the host cell. Most L. pneumophila effector proteins have not been characterized in detail, and their activities and host targets remain unknown. Interference with T4SS activity renders L. pneumophila avirulent, underscoring the important role of the translocated effectors for infection. Hence, a major focus of our research is to obtain in-depth insight into their biological functions. Over the past funding period, we have made important progress in developing and applying new research tools to decipher the biological role of effectors. We revealed that during infection L. pneumophila translocates several effectors that mimic host cell proteins with E3 ubiquitin ligase activity. E3 ubiquitin ligases catalyze the final step in an enzymatic cascade that results in the transfer of the small protein ubiquitin from E2 ubiquitin-conjugating enzymes to a particular target protein. Poly-ubiquitination of target proteins alters their cellular fate, often resulting in their proteasomal degradation. By encoding its own E3 ligases, L. pneumophila can hijack the host cell ubiquitination machinery and use it for its own benefit. We found that one of the L. pneumophila effectors is an E3 ligase relic that has been acquired by Legionella early during evolution and that has been extensively modified since then to best fulfil its current role. Despite the diversification, the mode of E2 recognition and binding has been preserved, suggesting that virulence-critical protein features are less prone to evolutionary diversification and represent preferred targets for therapeutic intervention. We also participated in a collaborative project that investigated the interaction between the effector protein RidL and components of the host cell retromer, a protein complex involved in cargo sorting at endosomal compartments. We discovered that RidL hijacks the function of retromer by pretending to be a cellular protein, a fascinating example of molecular mimicry. Knowledge about this molecular mimicry will form the basis for the development of novel drugs specifically designed to interfere with this mimicry. A similarly fascinating example of molecular mimicry emerged from our studies of the Legionella protein LegK7 which, upon delivery into host cells, phosphorylates MOB1, a scaffold protein of the Hippo pathway. The Hippo pathway is highly conserved in eukaryotes and well known for its role in controlling development but also tumorigenesis. Yet, the fact that Legionella targets this pathway during infection suggests an additional role of this pathway in immunity. We found that LegK7 combines functional features of not just one but two separate kinases of the Hippo signaling pathway, thereby taking control of it and causing changes in gene expression that promote intracellular bacterial growth. Interference with the change in gene expression renders human cells less susceptible to Legionella growth, providing us with a new way to treat infections by this pathogen. Taken together, our work has opened several new avenues for the development of novel therapeutic approaches to treat Legionnaires' disease and related illnesses. Our findings also show that studying intracellular pathogens can teach us important lessons on how our very own cells function, thus holding the key to obtaining in-depth insight into congenital and acquired human diseases, including cancer.